The invention relates to novel polysaccharide derivatives, such as cellulose ethers, i.e. in particular aminoalkyltrialkylsilylcelluloses, their preparation and their use in a process for coating surfaces with defined molecular layers.
The immobilization of biomolecules on solid carriers plays a decisive role in a large number of modern analysis and separation techniques, such as affinity chromatography, bioreactor technique and, in particular, bio- and chemosensory analysis.
For example, a detection in immunological tests and hybridization tests is carried out by specific reaction of receptor molecules adsorbed on a solid substrate surface with the species to be determined. In particular in the field of the highly sensitive detection of individual DNA, RNA, antigen and/or protein molecules, it is very important that the molecules in question are bound specifically and firmly to surfaces, and that unspecific adsorption of molecules at these substrate surfaces is prevented.
Various processes for binding biomolecules on solid substrate surfaces are known.
A simple option is adsorptive immobilization in which the binding on a substrate surface is purely by adsorption via non-covalent interaction. This method has various disadvantages. Immobilization is limited to substrates whose surface properties permit adsorptive binding and ensure sufficient stability. Gaps in the biomolecule layer may be formed by incomplete coating or desorption processes. Finally, control of both orientation and the amount of receptor molecules is unsatisfactory, so that it is difficult to achieve a reproducible preparation.
To avoid these disadvantages, there has been a quest for covalent immobilization processes which allow biomolecules to be bound covalently to solid substrates via functional groups. These covalent processes are based on bifunctional bridge reagents reacting both with the substrate surface and with the biomolecule.
These immobilization processes, which frequently proceed in several steps, are very time-and material-consuming. A further disadvantage of these methods is the formation of inhomogeneous polymeric surface structures, for example of silane films generated in practice, in the presence of moisture; see, for example, Joachim Renken et al., Anal. Chem. 1996, 68, pp. 176 to 182 in xe2x80x9cMultifrequency Evaluation of Different Immunosorbents on Acoustic Plate Mode Sensorsxe2x80x9d.
A further technique for coupling biomolecules on solid substrate surfaces is the self-assembly (SA) technique (see, for example, Kevin L. Prime et al., J. Amer. Chem. Soc. 1993, 115, pp. 10714 to 10721 in xe2x80x9cAdsorption of Proteins onto Surfaces Containing End-Attached Oligo(ethylenoxide): A Model System Using Self-Assembled Monolayersxe2x80x9d). Here, stable films of organic substances are formed by spontaneous self-assembly of the molecules during adsorption on solid substrates. The best-known SA systems are organic disulphides and thiols on gold surfaces. This method has the disadvantage that it is limited to only a few types of substrate, such as metals or specific oxides.
Related to the SA technique is the Langmuir-Blodgett (LB) technique. If suitable substances are spread on a surface of water, they spread out to form a monomolecular film. With the aid of this technique developed by Langmuir and Blodgett, it is possible to transfer these monomolecular films to solid substrates (see Katharine B. Blodgett et al., Physical Review, Vol. 51, June 1937, pp. 964 to 982 in xe2x80x9cBuilt-Up Films of Barium Stearate and Their Optical Propertiesxe2x80x9d). This process involves a very limited input of time and material.
Particularly ordered and stable films are obtained by using so-called non-arnphiphilic Stab-Haar polymers (for example M. Schaub et al., Thin Solid Films, 210/211, 1992, pp. 397 to 400 in xe2x80x9cInvestigation of molecular superstructures of hairy rodlike polymers by X-ray reflectionxe2x80x9d).
During transfer to the solid substrate, the polymer rods orientate themselves in parallel to the dipping direction. A particularly high stability of the films can additionally be achieved by crosslinking alkene substituents present in the polymer in a [2+2]-cycloaddition with UV irradiation (Gerhard Wegner, Thin Solid Films, 216, 1992, pp. 105 to 116 in xe2x80x9cUltrathin Films of polymers: architecture, characterization and propertiesxe2x80x9d).
Cellulose derivatives having olefinic side chains have been used with particular success. After the film has been transferred to the substrate, these can be converted in a modified Lemieux oxidation into carbonyl groups to which biomolecules are coupled covalently as xe2x80x9cSchiff basesxe2x80x9d (WO-A 95/08770 or Frank Lxc3x6scher et al., Proc. SPIE Vol. 2928, 1996, pp. 209 to 219).
By varying the length of time of exposure, it is additionally possible to vary the density of coverage. It is possible to couple biomolecules covalently to other cellulose derivatives having free amino or hydroxyl groups by using a bifunctional bridge reagent such as cyanuric chloride (see reference above).
This process has the disadvantage that it is limited to hydrophobic or hydrophobicized substrate types. Thus, hydrophilic glass or quartz substrates, for example, have to be hydrophobicized in complicated wet-chemical steps with the aid of, for example, silane derivatives prior to coating with hydrophobic LB substances.
Accordingly, it is the object of the invention to provide a process and a chemical compound suitable for this process allowing the application of at least one molecule layer onto a wide variety of different surfaces, independently of the hydrophilicity of surfaces.
This object is achieved on the one hand by polysaccharide derivatives, in particular cellulose derivatives having a degree of polymerization of  greater than 5, preferably mixed cellulose ethers comprising a) at least one hydrophobic and b) at least one nitrogen-containing substituent.
In preferred embodiments, the mixed cellulose ethers have, as substituent a), a trialkylsilyl and, as substituent b), an aminoalkyl group, where the alkyl radical in particular in the substituent a) has 1 or 2 C atoms and in the substituent b) has 2 to 8 C atoms. Additionally, the polysaccharide derivative may also comprise c) at least one further substituent which carries a group which is crosslinkable photochemically, by a free-radical reaction or thermally.
According to the present invention, the preferred mixed cellulose ethers are to be understood as compounds in which the H of individual OH groups of the cellulose skeleton is replaced by organic or organosilyl groups, i.e. the atom directly adjacent to the O is a C or Si. Furthermore, this expression can also include derivatives which additionally carry further substituents (in particular at the O of the OH group), an example of such an additional substituent being the substituent c). In the actual molecules (as information base, see Lothar Brandt in Ullmann""s Encyclopedia of Industrial Chemistry, Vol. A5, 2nd edition, keyword xe2x80x9ccellulose ethersxe2x80x9d, p. 461 ff.), it is not necessary for each individual molecule unit (anhydroglucose unit) in the cellulose ether molecule to be substituted at one or more OH groups, but the designation of the compound refers to the entirety of the molecules or molecule units, i.e. designates an average value; in general, a maximum of 3 OH groups per molecule unit can be substituted. For the preparation and/or the behaviour of cellulose derivatives comprising the substituents a) or c) (but not b)), reference is made to the literature reference above by Frank Lxc3x6scher et al. and to Dieter Klemm et al., Z. Chem., 24 (1984), Vol. 2, p. 62 in xe2x80x9c4-Dimethylamino-pyridin-katalysierte Synthese von Celluloseestern xc3xcber organolxc3x6sliche Synthese von Celluloseestern xc3xcber organolxc3x6sliche Trimethylcellulosexe2x80x9d.
The object of the invention is furthermore achieved starting with the known process for immobilizing biomolecules on a coated sheet-like carrier material in which the biomolecules are attached at or in the coating. In this case, the process according to the invention is characterized in that the coated sheet-like carrier material comprises within or outside the coating at least one of the abovementioned polysaccharide derivatives, in particular one of the mixed cellulose ethers.
Below, preferred embodiments of this process are illustrated in more detail.
To a solid surface, at least one monomolecular layer of the polysaccharide derivative is applied, where the polysaccharide derivative comprises, as substituent a), preferably a hydrophobic substituent having alkyl, alkenyl, aryl, alkylsilyl, alkenesilyl and/or arylsilyl radicals, but also other substituents making possible a transfer to surfaces using the Langmuir-Blodgett (LB) and/or Langmuir-Blodgett-Schxc3xa4fer (LBS) technique.
This layer can be applied by incubation in a solution, by a self assembly (SA) process or, preferably, using the Langmuir-Blodgett or Langmuir-Blodgett-Schxc3xa4fer technique. The polysaccharide derivatives according to the invention can adhere both to hydrophilic and to hydrophobic surfaces. Thus, this class of substances can be applied and used as a surface-modifying film.
These layers are additionally stabilized by incorporating photopolymerizable or thermally polymerizable groups into the molecule(s), for example cinnamoyl groups, but also all other groups known in chemistry, since they stabilize the layer by crosslinking by polymerization before, during and after the transfer (the publications mentioned at the outset are expressly incorporated herein with their disclosure content).
The polymerizable groups can be attached here either at the abovementioned polysaccharide derivative or else be present in the form of a further molecule which is, mixed with the polysaccharide derivative, applied at or in the layer. The polymerization can take place within a monolayer; however, if a plurality of monolayers are present on top of each other, polymerization can also take place between molecules of the individual layers.
The polysaccharide derivatives according to the invention are used in various applications. Thus, they can be employed as xe2x80x9cadhesion promotersxe2x80x9d which are located between the surface of the carrier material and one or more other layers. Further layers can be applied using all known methods, but they are preferably applied using the Langmuir-Blodgett or Langmuir-Blodgett-Schxc3xa4fer technique. Suitable further layers are, in particular, non-amp hiphilic Haar-Stab polymers, especially polysaccharide-containing molecules, such as trialkylsilylcellulose itself, for example trimethylsilylcellulose cinnamoate, but also other derivatives.
A particularly important application of these, multicomponent layers is the use for immobilizing molecules at these surfaces. Here, at least the upper-most layer applied to the first layer acting as xe2x80x9cadhesion promoterxe2x80x9d has functional groups which allow other molecules to be bound covalently. These include, for example, amino groups, aldehyde groups, carboxylic acid derivatives and/or groups which can be converted into active groups, for example olefinic double bonds, cinnamic acid derivatives and the like.
Furthermore, this substance class is also suitable for direct coupling of molecules, owing to the aminoalkyl groups present. The aminoalkyl groups serve as nucleophilic agent and form covalent bonds with molecules carrying electrophilic groups.
If the molecules of the substance class mentioned have silyl groups, for example trialkyl-, triaryl- or trialkenylsilyl groups, the surface properties can be modified such that the silyl groups can be removed after coating, leaving hydroxyl groups behind. This can be effected, for example, by action of acid.
To summarize the embodiments of the process according to the invention which are preferred in each case
the polysaccharide derivative is crosslinked before, during or after the coating is carried out,
the coating is crosslinked, as a whole or in individual layers, by addition of a crosslinking agent,
the coating comprises one or more individual layers,
polysaccharide derivative is present in all layers outside the sheet-like carrier material,
the layer comprising the polysaccharide derivative is the only coating,
the layer comprising the polysaccharide derivative is an intermediate layer between the sheet-like carrier material and the coating, where the coating, if appropriate, also comprises polysaccharide derivative,
biomolecules are attached to the polysaccharide derivative and
the substituent b) of the polysaccharide derivative is removed after coating has been completed, and OH groups are re-established.
Very particular preference according to the invention is given to a process for immobilizing biomolecules on a carrier material in which the biomolecules are attached at or in the coating and the coating is carried out by LB, LBS or SA techniques, giving photochemically crosslinkable and non-amphiphilic molecules where the sheet-like carrier material comprises within or outside the coating at least one mixed cellulose ether according to the description above.
The mixed cellulose ethers according to the invention can be prepared in such a way that a solution of trialkylsilylcellulose in an organic solvent is admixed with a reactive aminoalkane derivative which is insoluble or only sparingly soluble in this solvent, the reaction is carried out and the end product is isolated.